PROJECT SUMMARY Cardiac mesenchymal stem cells (C-MSC) are a unique pool of stem cells residing in the heart that play an important role in vascular homeostasis and physiological vascular cell turnover. Transplanting C-MSC into the heart has shown promise for vessel repair and angiogenesis, but poor survival of transplanted cells poses a major technical challenge. We reported that hypoxic preconditioning (HP) improves donor stem cell survival and angiogenesis in a HIF-1?- dependent manner. Mechanistically, C-MSC responses to HP correlate with the level of activation of Notch signaling, a cell-cell contact and pathway in stem cells that also mediates vascular smooth muscle cell (VSMC) differentiation of C-MSC. Moreover, we have identified a Notch-regulated microRNA, miR-322, the rodent homolog of human miR-424, which was reported to promote angiogenesis by blocking degradation of HIF-1? isoforms in human endothelial cells during hypoxia, suggesting a novel mechanism of crosstalk between Notch- regulated miR-322 and HIF-1?. We propose to investigate how Notch-1 and the newly identified Notch-1 target miR-322 sustain and potentiate the beneficial effects of HP on the vascular cell survival and angiogenic activity of stem cells. We will also determine whether harnessing these regulatory mechanisms in stem cells can enhance vessel protection and repair in a mouse model of myocardial infarction (MI). There are three aims: Aim 1: Test the hypothesis that Notch signaling regulates the beneficial effects of HP in stem cell-mediated vascular repair. Aim 2: Test the hypothesis that miR-322 mediates crosstalk between Notch1 and HIF-1? signaling in stem cells to enhance their activity. Aim 3: Test the therapeutic potential of targeting the Notch1/miR-322 axis to enhance stem cell-mediated vascular repair and angiogenesis in a mouse model of MI. Successful completion of the proposed studies will elucidate novel mechanisms associated with C-MSC mediated vascular repair and angiogenesis and enhance the efficacy of C-MSC therapy.